Our overall goal of this project is to develop a next-generation nanoparticle for targeted treatment of hepatocellular carcinoma (HCC). Liver cancer, including HCC, has the sixth highest cancer incidence and is the second leading cause of cancer mortality worldwide. While surgery or liver transplantation can be curative, most patients present with invasive HCC and are only eligible for palliative and locoregional treatments. Broadly cytotoxic treatments, including chemotherapy and radiation, have off-target toxicities to healthy hepatocytes, which is particularly dangerous to HCC patients due to a high incidence of underlying liver disease. Therefore, there is a critical need for a potent and highly targeted treatment option for liver cancer. To this end, we propose to develop a highly targeted nanoparticle gene delivery strategy for HCC tumors. Our preliminary data suggests that the biodegradable polymeric nanocarrier PBAE 536 can be used to selectively transfect HCC cells over healthy hepatocytes in vitro. Tumor-specific delivery of a therapeutic gene would enable targeted cancer cell killing with reduced off-target cytotoxicity to the surrounding healthy liver tissue. However, the therapeutic potential of this targeted nanotechnology has yet to be realized. The progress of clinical development is dependent on 1) optimization of nanoparticles for in vivo use and 2) the development of a safe, effective therapeutic DNA which is compatible with this delivery system. In this proposal, we will address both hurdles and test the final therapeutic in a preclinical model of human HCC. In Specific Aim 1, we will formulate and evaluate PBAE nanoparticles for gene delivery to orthotopic murine liver tumors. Nanoparticles will be systemically administered by intravenous injection, and off-target delivery and transfection will also be assessed. In Specific Aim 2, we will develop of a theranostic DNA vector targeted to HCC. We will use a thymidine kinase suicide gene (SR39), a dual-functional gene product that (1) converts the prodrug ganciclovir into a cancer-killing compound and (2) phosphorylates radiolabeled nucleoside analogs to enable PET imaging of gene expression. To enhance tumor targeting, we will employ ?-fetoprotein (AFP) transcriptional targeting, which we expect will restrict SR39 expression to HCC cells. Engineered AFP- SR39 vectors will be assessed in vitro for therapeutic gene expression in HCC cells and off-target healthy cell types. In Specific Aim 3, we will combine PBAE nanoparticles with our novel transcriptionally targeted theranostic plasmid for testing in a murine xenograft liver tumor model. We hypothesize that systemically administered PBAE nanoparticles will enable SR39 delivery to HCC tumors for safe and effective cancer killing and molecular genetic imaging. If successful, this interdisciplinary project will result in a novel precision approach to HCC treatment which may be adapted for clinical use.